Self-assembly of peptide-amphiphile nanofibers under physiological conditions

ABSTRACT

Peptide amphiphile compounds, compositions and methods for self-assembly or nanofibrous network formation under neutral or physiological conditions.

This application is a divisional of U.S. patent application Ser. No.10/368,517, filed Feb. 18, 2003, now U.S. Pat. No. 7,371,719, whichclaims priority from U.S. provisional application Ser. No. 60/357,228filed Feb. 15, 2002, both of which are incorporated herein in theirentireties by reference.

This invention was made with government support under Grant NumberDMR-9996253 awarded by the National Science Foundation, and Grant NumberF49620-00-1-0283/P01 awarded by the Air Force Office of ScientificResearch (MURI), and Grant Number DE-FG02-00ER45810 awarded by theDepartment of Energy. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Self-assembly and biomineralization are used for fabrication of manycomposite materials. Natural bone tissue is a particularly complexexample of such a composite with multiple levels of hierarchicalorganization (S. Weiner, H. D. Wagner, Annu. Rev. Mater. Sci. 28,271-298 (1998)). At the lowest level of this hierarchy is theorganization of collagen fibrils with respect to hydroxyapatite (HA)crystals. Collagen fibrils are formed by self-assembly of collagentriple helices while the HA crystals grow within these fibrils in such away that their c-axes are oriented along the long axes of the fibrils(W. Traub, S. Weiner, Proc. Nat. Acad. Sci. 86, 9822-9826 (1989)). Thepreparation of any material with structure on the nanoscale is achallenging problem. Fabrication of materials that resemble bone, evenat the lowest level of hierarchical organization, is even more difficultbecause it involves two dissimilar organic and inorganic nanophases thathave a specific spatial relationship with respect to one another. Oneapproach, using an artificial system, has been to prepare an organicnanophase designed to exert control over crystal nucleation and growthof the inorganic component.

The controlled nucleation and growth of crystals from organic templateshas been demonstrated in in vitro experiments and in a number of naturalbiomineralizing systems (S. Mann, J. P. Hannington, R. J. P. Williams,Nature 324, 565-567 (1986); D. D. Archibald, S. Mann, Nature 364,430-433 (1993); S. L. Burkett, S. Mann, Chem. Commun. 321-322 (1996); S.I. Stupp, P. V. Braun, Science 277, 1242-1248 (1997); J. Aizenberg, A.J. Black, G. M. Whitesides, Nature 398, 495-498 (1999); S. R. Whaley, D.S. English, E. L. Hu, P. F. Barbara, A. M. Belcher, Nature 405, 665-668.(2000); L. Addadi, S. Weiner, Angew. Chem., Int. Ed. Engl. 31, 153-169(1992); S. Mann, J. Chem. Soc., Dalton Tran. 3953-3961 (1997); S.Weiner, L. Addadi, J. Mater. Chem. 7, 689-702 (1997)). These studies ontemplated crystal growth suggest that nucleation occurs on surfacesexposing repetitive patterns of anionic groups. Anionic groups tend toconcentrate the inorganic cations creating local supersaturationfollowed by oriented nucleation of the crystal. Many groups haveinvestigated the preparation of bone-like materials using threedimensional organic substrates such as poly(lactic acid), reconstitutedcollagen and many others, and some studies shows a similar correlationbetween the crystallographic orientation of hydroxyapatite when theorganic scaffold is made from reconstituted collagen (G. K. Hunter, H.A. Goldberg, Biochem. J. 302, 175-179 (1994); G. M. Bond, R. H. Richman,W. P. McNaughton, J. Mater. Eng. Perform. 4, 334-345 (1995); J. H.Bradt, M. Mertig, A. Teresiak, W. Pompe, Chem. Mater. 11, 2694-2701(1999); N. Ignjatovic, S. Tomic, M. Dakic, M. Miljkovic, M. Plavsic, D.Uskokovic, Biomaterials 20, 809-816 (1999); F. Miyaji, H. M. Kim, S.Handa, T. Kokubo, T. Nakamura, Biomaterials 20, 913-919 (1999); H. K.Varma, Y. Yokogawa, F. F. Espinosa, Y. Kawamoto, K. Nishizawa, F.Nagata, T. Kameyama, J. Mater. Sci.: Mater. Med. 10, 395-400 (1999); A.Bigi, E. Boanini, S. Panzavolta, N. Roveri, Biomacromolecules 1, 752-756(2001); M. Kikuchi, S. Itoh, S. Ichinose, K. Shinomiya, J. Tanaka,Biomaterials 22, 1705-1711 (2001)). However, such results have neverbeen demonstrated in a pre-designed and engineered self-assemblingmolecular system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. In accordance with this invention: a) Chemical structure of apreferred peptide amphiphile, highlighting one or more structuralfeatures thereof. Region 1 may comprise a long alkyl tail that conveyshydrophobic character to the molecule and combined with the peptideregion makes the molecule amphiphilic. Region 2 may comprise one or more(four consecutive, shown) cysteine residues which when oxidized may formdisulfide bonds to provide a desired robust self-assembled structure.Region 3 may comprise a flexible linker region of one or more glycineresidues, preferably three, or functionally similar such residues ormonomers, to provide the hydrophilic head group flexibility from themore rigid crosslinked region. Region 4 may comprise a singlephosphorylated serine residue which is designed to interact stronglywith calcium ions and help direct mineralization of hydroxyapatite.Region 5 may comprise cell adhesion ligand Arg-Gly-Asp (RGD). b)Molecular model of an illustrated PA showing the overall conical shapeof the molecule going from the narrow hydrophobic tail to the bulkierpeptide region. c) Schematic showing the self-assembly of PA moleculesinto a cylindrical micelle.

FIGS. 2 a-2 d. a) Negative stain (phosphotungstic acid) transmissionelectron microscopy (TEM) of self-assembled nanofibers before covalentcapture. Fibers are arranged in ribbon-like parallel arrays. b) Vitreousice cryo-TEM of the fibers reveals the diameter of the fibers in theirnative hydrated state to be 7.6±1 nm. c) Positive stain (uranyl acetate)TEM of the self-assembled nanofibers after oxidative cross-linkingshowing electron dense regions due to the stain that localized on theperiphery of the fibers. d) Thin section TEM of positively stained(uranyl acetate) nanofibers after oxidative cross-linking and embeddingin epoxy resin. Two fibers are observed in cross-section (arrows)clearly showing the lack of staining in the interior of the fiber.

FIGS. 3 a-3 f. a) TEM micrographs of the unstained, cross-linkedpeptide-amphiphile fibers incubated for 10 min in CaCl₂ and Na₂HPO₄solution. The fibers arranged in bundles are visible due to the highconcentration of inorganic ions on their surface. b) After 20 minutesforming HA crystals (arrows) are observed in parallel arrays on some ofthe PA fibers. c) After 30 minutes mature HA crystals (arrows)completely cover the PA fibers. d) Electron diffraction pattern takenfrom a mineralized bundle of PA fibers after 30 minutes of exposure tocalcium and phosphate. The presence and orientation of the diffractionarcs corresponding to the 002 and 004 planes indicate preferentialalignment of the crystals with their c-axes along the long axis of thebundle. e) Plot of intensity versus inverse angstroms reveals that the002 and 004 peaks of hydroxyapatite are strongly enhanced along thepeptide-amphiphile fiber axis. f) Energy dispersive X-ray spectroscopy(EDS) profile of mineral crystals after 30 minutes of incubation revealsa Ca/P ratio of 1.67+/−0.08 as expected for HA.

FIG. 4. Scheme showing possible relationships between peptide-amphiphilefibers and hydroxyapatite crystals in the mineralized bundle. Arrowindicates the direction of the c-axes of the crystals.

FIG. 5. A tilt pair taken from mineralized PA fibers after 30 minutes ofincubation with calcium and phosphate demonstrating the plate shape ofthe crystals. The crystals that were “edge-on” (electron dense, narrowobjects) in the zero degree image lose contrast in the 45 degree rotatedimage (arrow 1) while the contrast of the crystals that were “face-on”in the zero degree images increase (arrow 2).

FIGS. 6 a-6 b. a) Nonphosphorylated PA fibers after 20 minutes ofincubation with calcium and phosphate shows only amorphous mineraldeposit concentrate on the fibers. b) Nonphosphorylated PA after 30minutes of incubation with calcium and phosphate continue to show onlyamorphous mineral in contrast with phosphorylated PA which shows heavycrystallization at this time point.

FIGS. 7-9. TEM micrographs for several cylindrical micelles preparedfrom PA molecules listed in Table 1. Specifically: FIG. 7, Top: Molecule#4 containing a C10 alkyl tail. Bottom: Molecule #13 containing a C22alkyl tail; FIG. 8, Top: Molecule 8 utilizing a tetra alanine sequencein place of tetra cysteine and containing a C16 alkyl tail. Bottom:Molecule 9 utilizing a tetra alanine sequence in place of tetra cysteineand containing a C10 alkyl tail; and FIG. 9, peptide-amphiphiles withthree different peptide head groups. Top: Molecule 10 with “KGE”.Middle: Molecule 14 lacking the phosphoserine group. Bottom: Molecule 15with (SEQ ID NO: 1) “IKVAV.”

FIGS. 10 a-10 b. Chemical structures of PA compositions 21 and 22, withreference to Table 2, below.

FIGS. 10C-E. Chemical structures of four peptide-amphiphiles used forself-assembly (SEQ ID NOS 23, 22 & 17 are disclosed respectively inorder of appearance).

FIG. 11. Molecule 1 self-assemblies into nanofibers upon drying from asolution at physiological pH.

FIGS. 12A-12E. TEM micrographs of positively stained peptide-amphiphilegels formed by addition of: A) Ca²⁺ to molecule 2 solution; B) Cd²⁺ andmolecule 2 solution; C) Ca²⁺ to molecule 4 solution; D) Fe²⁺ to molecule1 solution; and E) Zn²⁺ to molecule 1 solution.

FIG. 13. Self-assembly induced by mixing two different peptideamphipiles (21 and 22) containing opposite charges.

FIGS. 14A-C. TEM images of three different self-assembledpeptide-amphiphile nanofibers. 14A: Negatively charged peptideamphiphile 25 assembled with acid. 14B: Positively charged 24 assembledwith base. 14C: Nanofibers formed at neutral pH with a mixture of 24 and25.

FIGS. 15A-C. FT-IR spectra of the peptide-amphiphile gels. A. Thefragment of the spectrum of CaCl₂ induced gel of the molecule 27,showing the regions of Amide A, Amide I and Amide II bands. B. The AmideI and II region of the normalized spectra of the gels of the molecule 27assembled by addition of CaCl₂ and at low pH. C. The Amide I and IIregion of the normalized spectra of the gels of the molecule 32assembled by addition of CaCl₂, pH and KCl.

SUMMARY OF THE INVENTION

In light of the foregoing, it is an object of the present invention toprovide a nanostructured fiber-like system, or nanostructure providingother shapes such as spherical or oblate, and/or molecular componentsthereof, together with various methods for assembly and use to recreateor mimic the structural and/or functional interaction between collagenfibrils and hydroxyapatite crystals in bone or an extracellular matrix,thereby providing a nanostructured approach divergent from the priorart. It will be understood by those skilled in the art that one or moreaspects of this invention can meet certain objectives, while one or moreother aspects can meet certain other objectives. Each objective may notapply equally, in all its respects, to every aspect of this invention.As such, the following objects can be viewed in the alternative withrespect to any one aspect of this invention.

It is an object of the present invention to provide a composition whichcan be used for assembly of a molecular structure having dimensional andfunctional characteristics biomimetic with collagen fibrils.

It can also be an object of the present invention to provide ananostructured fibrous system as a template for tissue development.

It can also be an object of the present invention to provide a compositeof a mineralized nanofiber structure biomimetic with collagen fibrilsand hydroxyapatite crystals in natural bone tissue.

It can also be an object of the present invention to provide a systemfor the facile self-assembly of nanostructured fibers under a particularpH regime or under substantially neutral or physiological pH conditions,for use in conjunction with one or more of the preceding objectives,such fibers as can be reversibly stabilized to promote structuralintegrity.

It can also be an object of the present invention to provide peptideamphiphile compositions comprising two or more oppositely chargedpeptide components, each such component as can further include the sameor a differing bioactive epitope sequence, for subsequent biomedicalapplications including without limitation either in vitro or in vivodrug delivery, cell therapies or tissue engineering.

It can also be an object of the present invention, irrespective of anyend use application, to provide one or more peptide amphiphilecompositions which are stable at physiological pHs with or withoutcovalent cross-linking, such stability of as can be provided via ioniccross-linking of charged functional groups present within the componentamphiphiles of such compositions.

It can also be an object of the present invention to provide amethodology using fibers in a stabilized three-dimensional structure todirect and/or control mineralization and crystal growth thereon.

It can also be an object of the present invention to provide a molecularsystem for the design and engineering of specific nanofibers andcomponents thereof to target particular cell and/or mineral growth enroute to a variety of hard or soft biomimetic materials for biologicaland non-biological applications, the later including, but not limitedto, catalysis, photonics and electronics.

Other objects, features, benefits and advantages of the presentinvention will be apparent from this summary and its descriptions ofvarious preferred embodiments, and will be readily apparent to thoseskilled in the art having knowledge of natural biomineralizing systems.Such objects, features, benefits and advantages will be apparent fromthe above as taken into conjunction with the accompanying examples,data, figures and all reasonable inferences to be drawn therefrom.

With respect to various embodiments, the present invention comprises useof self-assembly techniques, such self-assembly as may be employed inconjunction with mineralization to prepare a nanostructured compositematerial which recreates or mimics the structural orientation andinteraction between collagen and hydroxyapatite observed in bone. Acomposite may be prepared by self-assembly, covalent capture, andmineralization of one or more peptide-amphiphile (PA) compositions. Asevident from the preceding, the peptide-amphiphile (PA) compositions ofthis invention can be synthesized using preparatory techniqueswell-known to those skilled in the art—preferably, by standard solidphase chemistry, with alkylation or other modification of the N-terminusof the peptide component with a hydrophobic moiety. Mono or di-alkylmoieties attached to the N or C termini of peptides may influence theiraggregation and secondary structure in water in both synthetic andnatural systems. As illustrated in several embodiments, a hydrophobic,hydrocarbon and/or alkyl tail component with a sufficient number ofcarbon atoms coupled to an ionic peptide having a preference forbeta-strand conformations can in certain embodiments be used to createan amphiphile that assembles in water into nanofiber structures. Theamphiphile's overall conical shape can also have an effect on suchassemblies. (J. N. Israclachvili Intermolecular and surface forces; 2nded.; Academic: London San Diego, 1992). The hydrophobic tails pack inthe center of the assembly with the peptide segments exposed to anaqueous or hydrophilic environment. These cylindrical nanostructures canbe viewed as fibers in which the chemistry of the peptide region isrepetitively displayed on their surface. Comparably, consistent withthis invention, amphiphile molecules can also be designed to providemicelles having structural shapes that may differ from a fiber likeappearance.

Without limitation, three structural and/or functional features can beengineered into the peptide region of a PA composition of thisinvention. First, the prepared fibers are optimally robust and, for thisreason, one or more cysteine amino acid residues—four in someembodiments and/or, optionally, consecutive—can be incorporated in thesequence for covalent capture of supramolecular nanofibers. (D. Y.Jackson, D. S. King, J. Chmielewski, S. Singh, P. G. Schultz, J. Am.Chem. Soc. 113, 9391-9392 (1991); T. D. Clark, K. Kobayashi, M. R.Ghadiri, Chem Eur J 5, 782-792 (1999); Y. Y. Won, H. T. Davis, F. S.Bates, Science 283, 960-963 (1999); E. R. Zubarev, M. U. Pralle, L. M.Li, S. I. Stupp, Science 283, 523-526 (1999); E. A. Archer, N. T.Goldberg, V. Lynch, J Am Chem Soc 122, 5006-5007 (2000); F. Cardullo, M.C. Calama, B. H. M. Snellink-Ruel, J. L. Weidmann, A. Bielejewska, R.Fokkens, N. M. M. Nibbering, P. Timmerman, D. N. Reinhoudt, Chem. Comm.5, 367-368 (2000)). Such residues can be used to form disulfide bondsbetween adjacent PA molecules upon oxidation to lock the supramolecularstructure into place. The formation of the disulfide bonds isreversible, as described elsewhere herein, allowing self correction ofimproper disulfide bonds or return to the supramolecular structure bytreatment with mild reducing agents.

With regard to a second feature, the fibers of various embodiments maybe able to nucleate the formation of HA crystals in the properenvironment. It is well known that acidic moieties play a key role inbiomineralization processes and in the formation of calcium phosphateminerals phosphorylated groups are particularly important. (G. K.Hunter, H. A. Goldberg, Biochem. J. 302, 175-179 (1994); S. Weiner, L.Addadi, J. Mater. Chem. 7, 689-702 (1997)). For example in dentin, thephosphosphoryn protein family contains numerous repeats of the sequencesAsp-Ser(P)-Ser(P) and Ser(P)-Asp (A. George, L. Bannon, B. Sabsay, J. W.Dillon, J. Malone, A. Veis, N. A. Jenkins, D. J. Gilbert, N. G.Copeland, J. Biol. Chem. 271, 32869-32873 (1996)). These massivelyphosphorylated proteins are closely associated with the collagenextracellular matrix (ECM) and are known to play an important role in HAmineralization (A. Veis In Biomineralization: Chemical and BiologicalPerspectives; S. Mann, J. Webb, J. R. P. Williams, Eds.; VCH: WeinheimN.Y., 1989; pp 189-222). Accordingly, at least one phosphoserine residuecan be incorporated into the peptide sequence which, after selfassembly, allows the fiber to display a highly phosphorylated surfacefunctionally biomimetic to a long peptide segment. This, in part, may beused to simulate a repetitive organization of phosphate groups found inphosphosphoryn proteins.

Third, with respect to one or more of the preceding embodiments orothers within the scope of this invention, it would be beneficial forbiomedical applications to provide fibers promoting surface adhesion andgrowth of cells. Another collagen associated protein, fibronectin,contains the sequence Arg-Gly-Asp (RGD). As this sequence has been foundto play an important role in integrin-mediated cell adhesion, an RGDsequence can also be included in preferred peptide components and/or PAcompositions, depending upon end-use application. Collectively, theseand other design principles led to preparation of a PA molecule of thetype shown in FIG. 1. While the PA compound of FIG. 1 is shown with abioactive RGD sequence, other epitope sequences can be used, asdescribed elsewhere herein including, but not limited to, the IKVAV (SEQID NO: 1) sequence.

Notwithstanding the numerous embodiments provided above, broader aspectsof the present invention include a peptide amphiphilecompound/composition having 1) a hydrophobic component and 2) a peptideor peptide-like component further including a bioactive epitopesequence. In various preferred embodiments, the hydrophobic component ofsuch a compound or composition is of sufficient length to provideamphiphilic behavior and nanofiber assembly/formation in water oranother polar solvent system. Typically, such a component may be about aC₆ or greater hydrocarbon moiety, although other hydrophobic,hydrocarbon and/or alkyl components could be used as would be well-knownto those skilled in the art to provide similar structural or functionaleffect. Such hydrophobic components include, without limitation,cholesterol, biphenyl and p-aminobenzoic acid. Regardless, a peptidecomponent of such a composition may include the aforementioned RGDsequence found especially useful for the nanofiber cell adhesion andmineralization described herein. Alternatively, an IKVAV (SEQ ID NO: 1)sequence can be incorporated into a PA compound.

Preferred peptide components of such compounds/compositions can alsoinclude a phosphoryl-functionalized residue or sequence (as indicatedwith the corresponding letter code and a parenthetical “P”), asdescribed above. Inclusion of a phosphoserine residue, S(P), has beenfound especially useful for HA mineralization. Other embodiments caninclude, for example and without limitation, a phosphotyrosine residue.The peptide component of such compositions can also include a residue orsequence capable of promoting intermolecular bonding and structuralstability of the micelles/nanofibers available from such compositions. Asequence of cysteine residues can be used with good effect, providingfor the facile intermolecular oxidation/reduction of the associatedthiol functionalities.

Peptide components of this invention preferably comprisenaturally-occurring amino acids. However, incorporation of knownartificial amino acids such as beta or gamma amino acids and thosecontaining non-natural side chains, and/or other similar monomers suchas hydroxyacids are also contemplated, with the effect that thecorresponding component is peptide-like in this respect. One examplealready tested includes an amino acid substituted with a thiophenemoiety so that polymerization can produce electrically conductive and/orfluorescent materials. Accordingly, such artificial amino acids,hydroxyacids or related monomers can be used to meet the spacer,phosphorylation and/or intermolecular bonding objectives describedabove.

Various aspects of the present invention can be described with referenceto the peptide amphiphile illustrated in FIG. 1, but consistent withbroader aspects of this invention, other compounds and compositions canbe prepared in accordance with this invention and used for theself-assembly of fibrous cylindrical micelles and correspondingnanostructures. See, Table 1, below.

TABLE 1 SEQ ID PA NO: N-terminus Peptide (N to C) C-terminus 1 2 C16CCCCGGGS(P)RGD H 2 3 C16 CCCCGGGS(P) H 3 4 H CCCCGGGS(P)RGD H 4 5 C10CCCCGGGS(P)RGD H 5 6 C6 CCCCGGGS(P)RGD H 6 7 C10 GGGS(P)RGD H 7 8 C16GGGS(P)RGD H 8 9 C16 AAAAGGGS(P)RGD H 9 10 C10 AAAAGGGS(P)RGD H 10 11C16 CCCCGGGS(P)KGE H 11 12 C10 AAAAGGGS(P)KGE H 12 13 C16 AAAAGGGS(P)KGEH 13 14 C22 CCCCGGGS(P)RGD H 14 15 C16 CCCCGGGSRGD H 15 16 C16CCCCGGGEIKVAV H 16 17 C16 CCCCGGGS(P)RGDS H 17 18 C_(n) LLLKK-X H 18C_(n) LSL-X H 19 19 C_(n) LSLS-X H

Depending upon desired cell or mineral growth, a phosphorylated moietyor residue may not be included (see PAs 14 and 15). As discussed above,cellular adhesion or other bio-interaction may be promoted by aparticular sequence of the peptide component. With reference to PAs10-12 and 15, a non-RGD sequence can be utilized depending upon cellulartarget or end-use application. In particular, the IKVAV (SEQ ID NO: 1)sequence has been identified in other contexts as important for neurongrowth and development. The YIGSR (SEQ ID NO: 20) sequence, as discussedmore fully below, can also be used. Accordingly, the amphiphilecompositions of this invention can include a peptide component havingsuch a sequence for corresponding use. Other residues and/or bioactiveepitope sequences capable of promoting cell adhesion, growth and/ordevelopment are known in the art and can be used in conjunction with thepresent invention, such as residues/sequences as can be incorporatedinto the peptide components and/or PA compositions of this inventionusing available synthetic techniques or straight-forward modificationsthereof. With respect to drug delivery and related end-use applications,various bioactive epitope sequences incorporated into the PAcompounds/compositions of this invention can be used to adsorb, bindand/or deliver a number of hydrophilic therapeutic agents, including butnot limited to growth factors, related co-factors and/or activators.Conversely, such compound/compositions can be used to control thedelivery rate of various hydrophobic/hydrocarbon therapeutic agentsbound and/or encapsulated within the hydrophobic components thereof.With respect to Table 1, it is noted that several PAcompounds/compositions do not include cysteine residues: while such aresidue or peptide sequence can be used to enhance intermolecularnanofiber stability, it is not required for micelle formation in thefirst instance. Reference is made to FIGS. 7-9 for TEM micrographs ofseveral PA compositions identified in Table 1.

Further reference is made to Table 1 and, in particular, PA compositions17-19. Various other embodiments of this invention may comprise theresidues shown, or where optionally X may further comprise one or moreof the aforementioned residues as may be utilized for intramolecularstructural flexibility, intermolecular stability, mineralization and/orcellular interaction. Accordingly, each such composition can optionallycomprise, as desired for end-use application, one or more glycine,cysteine, phosphorylated and/or cell growth, development or adhesionresidues. In accordance with the preceding discussion, the amphiphilicnature of such compositions provides for the presence of a suitablyhydrophobic component C_(n), where n is an integer corresponding to thenumber of carbon atoms in such a component sufficient to providesufficient amphiphilic character and/or assembly structure. Withoutlimitation, various embodiments of such compositions comprise ahydrophobic component (e.g., alkyl, biphenyl, cholesterol, etc.) ofabout C₆- about C₂₆. More specifically and without limitation, asequence such as that provided by PA composition 17 (or as furthercomprising residue(s) X) can be utilized to modify and/or enhance therate of nanostructure self-assembly. Likewise, without limitation, thesequences of PA compositions 18 and 19 (or as further comprisingresidue(s) X) can be used, as needed, to improve the structural ormechanical properties of the corresponding available nanostructured gelmaterials. Implementation of such peptides in the PA compositions ofthis invention do not, of course, preclude use of other residues. Forexample, PA compositions 17-19 can further comprise one or more glycine,cysteine, phosphorylated and/or cellular interaction residues, inaccordance with this invention.

In part, the present invention also provides a sol-gel systemincluding 1) a polar or aqueous solution and/or containing of one ormore of the amphiphile compounds or compositions described herein, and2) a factor or reagent sufficient to induce assembly, agglomeration ofgelation under neutral or physiological conditions. Such gelation and/orself-assembly of various PA compositions into micellular nanofibers canbe achieved under substantially neutral and/or physiological pHconditions through drying, introduction of a mono- or multivalent metalion and/or the combination of differently charged amphiphiles. Theapproach of using differently charged amphiphiles can also be utilizedto deliver in the self assembling nanofibrous system two or morebioactive molecules, each bearing different charges and this waycombining the gelation technology with the delivery of multiplebiological signals. Such facile factors, as described more fully belowand in several of the following examples, can extend the sol-gel systemand/or methodology of this invention to a variety of medicalapplications. These and other aspects of the present invention can bedescribed with reference to the PA compositions provided in Table 2,below, with further reference to FIGS. 1, 10A-B and Table 1, above.

TABLE 2 Net SEQ ID N- Peptide Charge at PA NO: terminus (N to C)C-terminus pH 7 17 2 C16 CCCCGGGS(P)RGD COOH −3 18 9 C16 AAAAGGGS(P)RGDCOOH −3 19 10 C10 AAAAGGGS(P)RGD COOH −3 20 15 C16 CCCCGGGSRGD COOH −121 16 C16 CCCCGGGEIKVAV COOH −1 22 21 C16 CCCCGGGKIKVAV CONH₂ +1 23 17C16 CCCCGGGS(P)RGDS COOH −3 24 22 C16 AAAAGGGKYIGSR CONH₂ +2 25 23 C16AAAAGGGEIKVAV COOH −1

The peptide epitopes on molecules 22-25 demonstrate the biomedicalpotential of the self assembling systems described here. RGD is the wellknown cell adhesion ligand found in fibronectin while IKVAV (SEQ IDNO: 1) and YIGSR (SEQ ID NO: 20) are laminin sequences known to interactwith mammalian neurons. IKVAV (SEQ ID NO: 1) promotes neurite outgrowthin mammalian neurons, while YIGSR (SEQ ID NO: 20) plays a related rolein neuronal cell-substrate adhesion. While these and other bioactiveepitope sequences can be used to effect cell adhesion, proliferation ordifferentiation and related outcomes, in a broader context, the PAcompounds/compositions and related methods of this invention can be usedin conjunction with any epitope sequence capable of cellular interactionand/or binding to a cellular membrane receptor. In particular, peptideamphiphiles 23 and 25 have a net negative charge at neutral pH, whereasPAs 22 and 24 have a net positive charge. Electrostatically drivenco-assembly between PA compounds 24 and 25 as well as 23 and 22 providemixed nanofibers that simultaneously present two biological signals in acellular environment.

Various peptide-amphiphile compositions are shown in Tables 1 and 2, butare provided only by way of illustrating one or more aspects of thisinvention. It will be understood by those skilled in the art that arange of other compositions are also contemplated. For example, thepeptide components can be varied, limited only by functionalconsiderations of the sort described herein. Accordingly, peptide lengthand/or sequence can be modified by variation in number or identity ofamino acid or substituted monomer. Further, it will be understood thatthe C-terminus of any such sequence can be construed in light of anassociated carboxylate functionality or derivative thereof. While theN-terminus of the peptides is illustrated with respect to the referencedconjugated hydrophobic and/or hydrocarbon components, it will beunderstood that such components can also be varied by length and/orcomposition, such variation limited only by the functionalconsiderations presented herein. For example, a sixteen carbon (C16)component can comprise but is not limited to a straight-chain alkylhydrocarbon. As would be understood by those skilled in the art, thestructure and/or chemistry of such a component can be varied with regardto a particular, desired functionality of an amphiphile composition orassembly thereof. Likewise, the length of such a component is limitedonly by way of the degree of hydrophobicity desired for a particularamphiphile composition and/or assembled structure, in conjunction with agiven solvent medium.

Consistent with broader aspects of this invention, it was found thatrepresentative peptide amphiphiles (for example, PAs 17-19 and 21, Table2 and those of Table 1), when dissolved at neutral pH were dried ontosurfaces, self-assembled into cylindrical micelles (FIG. 11). Such afacile approach allows this novel material to be formed in a pHindependent manner, as may be important in applications that areparticularly sensitive to changes in pH, including the delivery of cellsin conjunction with the gelling system and generally avoiding contactbetween tissues and materials at non-physiological pH.

Illustrating another such factor, the self-assembly of the PA molecules(for example, but not limited to PAs 17, 18 and 20, Table 2 and Table 3)into nanofibers can be induced by addition of metal ions such as, butnot limited to K⁺, Ca⁺², Mg⁺², Cd⁺², Fe⁺² and Zn⁺², and metal ionshaving higher oxidation states such as but not limited to the trivalentAl⁺³ and Fe⁺³. Self-supporting gels can be formed upon addition of astoichiometric excess of such ions, preferably on the order of about 2-3metal ions per molecule of PA. TEM study of these gels reveals a networkof nanofibers that pack into flat bundles, similar to those found in thegels self-assembled by acidification (FIG. 12). (See examples 19a-c andTable 3, below.)

More generally, such PA compositions can be prepared with appropriatealkaline, alkaline earth, transitional metal salts or reagentscomprising such salts. With further reference to Table 3, whilenegatively-charged PAs tend to gel in the presence of metal ions,gelation properties may vary depending upon PA composition and metal ionidentity. Self-assembly and/or gelation can also be varied throughmodification of the hydrophobic and hydrophilic portions/regions of thePA compositions, one relative to the other. With regard to thehydrophilic region, choice of amino acid residues can affect gelationdepending upon net charge of and/or charge distribution therein.

As yet another factor inducing gelation under physiological conditions,consider two amphiphiles, such as but not limited to PAs 21 and 22(Table 2), one having a net negative charge at neutral pH and one havinga net positive charge at neutral pH—both dissolved at neutral pH.However, upon mixing such amphiphiles immediately form a gel.Examination by negative stain TEM shows the gel composed of cylindricalmicelles (FIG. 13).

In one possible working model of the molecular organization of the mixedPA nanofibers, the hydrophobic alkyl tails are hidden in the center ofthe micelles with the more hydrophilic peptide segments of the moleculesin contact with the aqueous environment. The cylindrical structure ofthis micelle could in part be explained by the tapered shape ofindividual molecules, but a second driving force might be beta sheethydrogen bonding among peptide segments down the long axis of thefibers: the parallel β-sheet hydrogen bonding conformation is observedby FT-IR and on the unusual dominance of the cylindrical self-assemblymotif across such a broad concentration range (<about 0.001% to >about10% by weight). (See Example 9, below.)

In the case of the mixed PA fibers, two oppositely charged molecules arebelieved thoroughly mixed within any given nanofiber as opposed tomolecules segregating into mixtures of homomeric fibers. If homomericfibers formed in spite of the highly unfavorable charge concentrationsassociated with these structures, it would be expected that the fiberspack into bundles of oppositely charged fibers in order to reduceelectrostatic repulsion. However, the same amount or less bundling inthe mixed PA fibers is observed as compared to fibers formed from asingle PA molecule (FIG. 14).

Self-assembly and/or gelation under physiological conditions, as inducedby the preceding factors, raise numerous implications regarding end useapplication and effect. Without limitation, with reference to thepreceding, a binary or higher PA mixture makes available a sol-gelsystem for the formation of micellular nanofibers in a aqueousenvironment at neutral and/or physiological pH conditions. As discussedelsewhere herein, such a combination of two or more PA compounds can beused to assemble nanofibers with a range of residues providing acorresponding variety of concurrent chemical or biological signals forcell adhesion proliferation, differentiation and the like, yieldingenhanced properties with regard to tissue engineering or regenerativeapplications. Alone, or in conjunction with one or more of the otherfactors discussed herein, it is contemplated that preferred medical ortherapeutic embodiments of such a system or methodology can beimplemented upon step-wise introduction and mixing of the subject PAcompositions, with in situ gel formation.

Accordingly, such a system can be used in conjunction with a drug,medication or other therapeutic agent, as would be understood by thoseskilled in the art: the subject drug or therapeutic agent can beprovided with or introduced to an appropriate aqueous or polar mediumseparately or in conjunction with one or more PA compounds. Introductionof a reagent and/or factor induces nanofiber assembly and/or gelation,incorporating such a drug/agent therein, if hydrophobic, or as bound toor sorbed on the surface thereof, if hydrophilic. Disassembly orsolubilization of the nanofibrous network or gel can release or deliverthe drug/agent as or where required. As would be understood by thoseskilled in the art made aware of this invention, a range of bothhydrophobic and hydrophilic drugs/agents can be utilized herewith. Inparticular, with regard to the peptide epitopes thereof, hydrophilicgrowth factors, co-factors and/or activators can be adsorbed on,delivered with and/or released by the PA compounds/compositions of thisinvention.

In preferred embodiments, regardless of any such factor or physiologicalcondition, the amphiphile composition(s) of such a system includes apeptide component having residues capable of intermolecularcross-linking. The thiol moieties of cysteine residues can be used forintermolecular disulfide bond formation through introduction of asuitable oxidizing agent. Conversely, such bonds can be cleaved by areducing agent introduced to the system. The concentration of cysteineresidues could be varied to control the chemical and biologicalstability of the nanofibrous system and therefore control the rate ofdrug or therapeutic delivery using the nanofibers as the carriers.Furthermore, enzymes could be incorporated in the nanofibers to controlbiodegradation rate through hydrolysis of the disulfide bonds. Suchdegradation and/or the concentration of cysteine residues can beutilized in a variety of tissue engineering contexts.

Corresponding to one or more preferred embodiments of such a material,composition or system, the present invention can also include ananostructured template for mineral crystal and/or cellular growth. Sucha template includes a micelle assembly of amphiphile composition(s) ofthe type described herein, wherein the peptide component thereof mayinclude a residue or sequence capable of intermolecular bond formation.In preferred embodiments, as described above, cysteine residues can beused for intermolecular disulfide bond formation. Various otherpreferred embodiments can further include one or more phosphorylatedresidues to promote crystal growth and/or mineralization, depending upona desired material or tissue target.

In the context of biomimetic hard material or tissue, the presentinvention can also include an organo-mineral composite having ananostructured peptide amphiphile template with mineral crystalsthereon. As described above, this aspect of the present invention can beillustrated with the present amphiphile compositions, assembled asnanostructured fibers, used to nucleate and grow hydroxyapatitecrystals. In preferred embodiments, the amphiphilic compositions includepeptide components having one or more residues promoting crystalnucleation and growth. Such preferred peptide components can alsoinclude one or more residues capable of intermolecular bonding tostabilize the nanofiber template. While this inventive aspect has beendescribed in conjunction with hydroxyapatite nucleation and growth, themineral component of this composite can include other inorganiccompounds and/or oxides. Such residues, sequences or moieties are of thetype described herein, or as would otherwise be understood by thoseskilled in the art made aware of this invention.

Regardless, the c-axes of the mineral crystals of such composites arealigned with the longitudinal fiber axes, in a manner analogous to thealignment observed between collagen fibrils and HA crystals in naturalbone tissue. Accordingly, the present invention can also include amethod of using a peptide amphiphile, in accordance with this invention,to promote and control HA crystal growth. The identity of the PAcompositions used therewith is limited only by way of those structuralconsiderations described elsewhere herein. Consistent therewith and withthe broader aspects of this invention, such a method includes 1)providing an aqueous or other suitable polar medium of one or morepeptide amphiphile compositions, 2) inducing assembly thereof intocylindrical micelle structures, 3) optimally stabilizing the structureswith intermolecular bond formation, and 4) introducing to the mediumreagents suitable for the preparation of HA and crystalline growththereof on the nanofiber micelle structures.

As provided elsewhere herein, one or more amphiphile compositions can beused to provide fibers with a variety of cell adhesion, mineralizationand/or structural capabilities. A combination of such compositions canbe used to assemble a nanofibrous matrix with synergistic propertiesbeneficial for a particular crystal and/or cellular development, suchcompositions as may vary according to peptide component, residuesequence, hydrophobic or hydrocarbon component and/or resulting PAcompound or assembly configuration. As described elsewhere herein, acombination of charged PA compositions can be used to effectassembly—likewise with drying and incorporation of divalent cations.

EXAMPLES OF THE INVENTION

The following non-limiting examples and data illustrate various aspectsand features relating to the compositions, micelles, composites and/ormethods of the present invention, including self-assembly of apeptide-amphiphile nanofiber system, as is available through themethodologies described herein. In comparison with the prior art, thepresent structures, template designs and related methods provide resultsand data which are surprising, unexpected and contrary thereto. Whilethe utility of this invention is illustrated through the use of severalamphiphiles, biomimetic micelles and resulting organo-mineralcomposites, it will be understood by those skilled in the art thatcomparable results are obtainable with various other amphiphiles,nanofibers/micelles and/or composites, as are commensurate with thescope of this invention.

Example 1

After synthesis (see, example 10), the PA of FIG. 1 (characterized by ¹HNMR and matrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF MS): matrix ion [M-H]⁻¹=1333) was treated withdithiothreitol (DTT) at a pH of 8 to reduce all cysteine residues tofree thiols. At this pH the PA was found to be soluble in excess of 50mg/ml in water. However, upon acidification of the solution below pH 4the material rapidly becomes insoluble. Solutions more concentrated than2.5 mg/ml form birefringent gels in water that are self-supporting uponinversion of the container. Examination of the gels by cryo-TEM, whichpreserves the native, hydrated state of the material, revealed a networkof fibers with a diameter of 7.6±1 nm and lengths up to several microns(FIG. 2 b). Negatives were digitized in the Umax PowerLook scanner withresolution 1200 dpi. The average thickness of the PA fibers wasdetermined by two different procedures. First the Fourier transform ofthe images and following measurements of the distances between the peakswere performed in the NIH Image 1201.1262 program. The second approachapplied was averaging technique based on the cross-correlation method,widely used in single particle reconstruction using the EMAN 1201.1202program. For each data set 1200-1300 individual boxes were selected. (E.Beniash, W. Traub, A. Veis, S. Weiner, J. Struct. Biol. 132, 212-225(2000). Positively and negatively stained dried fibers were found tohave diameters of 6.0±1 nm (FIG. 2 a, c). TEM using the positive stainuranyl acetate, which preferentially stains acidic groups, revealedincreased electron density at the periphery of the fiber. (J. R. Harris,Ed. Electron Microscopy in Biology, A Practical Approach; OxfordUniversity Press: New York, 1991). Additionally, gels that were stained,embedded in epoxy resin and sectioned for TEM, showed fibers incross-section in which donut shaped patterns were observed indicatingstaining only on the outer portion of the fiber (FIG. 2 d). The twopositive staining experiments of this example indicate that thehydrophobic alkyl tails pack on the inside of the fiber and the acidicmoieties of the peptide are displayed on the surface of the fiber.

Example 2

The formation of the fibers was found to be concentration independentover more than three orders of magnitude (0.01 mg/ml to 50 mg/ml),however a second level of hierarchy was observed that was concentrationdependent. As the concentration of the PA was increased, a larger numberof the fibers were observed to pack into fiat ribbons of fibers (FIG. 2a,c).

Example 3

Examination of the self-assembled material by FT-IR revealed a bimodalamide I peak with maxima at 1658 cm⁻¹ (α-helix) and 1632 cm⁻¹ (β-sheet)along with a N—H stretching peak at 3287 cm⁻¹ indicating the formationof a hydrogen bonded structure, possibly utilizing a combination ofβ-sheet and α-helical secondary structure in the fibers (S. Krimm, J.Bandekar, Adv. Protein Chem. 38, 181-364 (1986); W. K. Surewicz, H. H.Mantsch, D. Chapman, Biochemistry 32, 389-394 (1993)). Based on theabove data the nanofibers were modeled as cylindrical micelles in whichthe alkyl tails pack on the inside of the fiber and peptide segments aredisplayed on the outside containing both β-sheet and α-helical secondarystructure. This model results in a fiber with a diameter of 8.5 nm,which is within the margin of error of our TEM measurements (FIG. 1 c).

With reference to examples 4-9, the following abbreviations are employedfor the reagents used. PA: peptide-amphiphile, TEM: transmissionelectron microscopy, DTT: dithiothreitol, EDT: ethanedithiol, TIS:triisopropyl silane, TFA: trifluoroacetic acid, HBTU: (2-(1h-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate,DiEA: Diisopropylethylamine. Except as noted below, all chemicals werepurchased from Fisher or Aldrich and used as provided. DiEA andPiperidine were redistilled before use. Amino acid derivatives,derivatized resins and HBTU were purchased from Nova Biochem. All waterused was deionized with a Millipore Milli-Q water purifier operating ata resistance of 18 MΩ.

Example 4

With reference to examples 5-9, below, the peptide-amphiphiles of Table2 were prepared on a 0.25 mmole scale using standard FMOC chemistry onan Applied Biosystems 733A automated peptide synthesizer. All peptidesprepared have a C-terminal carboxylic acid and were made usingpre-derivatized Wang resin. After the peptide portion of the moleculewas prepared the resin was removed from the automated synthesizer andthe N-terminus capped with a fatty acid containing 10 or 16 carbonatoms. The alkylation reaction was accomplished using 2 equivalents ofthe fatty acid, 2 equivalents HBTU and 6 equivalents of DiEA in DMF. Thereaction was allowed to proceed for at least six hours after which thereaction was monitored by ninhydrin. The alkylation reaction wasrepeated until the ninhydrin test was negative. In general the longerthe fatty acid the more repetitions were required to drive the reactionto completion.

Cleavage and deprotection of the peptide-amphiphiles containing cysteinewas done with a mixture of TFA, water, TIS and EDT in a ratio of91:3:3:3 for three hours at room temperature. The cleavage mixture andtwo subsequent TFA washings were filtered into a round bottom flask. Thesolution was roto-evaporated to a thick viscous solution. This solutionwas triturated with cold diethylether. The white precipitation wascollected by filtration, washed with copious cold ether and dried undervacuum. Typically, 200 mg of the peptide-amphiphile powder was dissolvedin 20 ml of water with the addition of 1M NaOH to adjust the pH of thesolution to 8 and 200 mg of dithiothreitol (DTT) to reduce all cysteineamino acids to the free thiol and allowed to stir overnight. Thesolution was then filtered through a 0.2 μm nylon Acros filter into anew round bottom flask. This 10 mg/ml (1% by weight) solution was usedfor all subsequent manipulations. Work up of peptide-amphiphiles notcontaining cysteine were performed as above accept that ethanedithiolwas omitted from the cleavage reaction and DTT was not used in thepreparation of aqueous solutions. Peptide-amphiphiles were characterizedby MALDI-TOF MS and were found to have the expected molecular weight.

Example 5

Nanofibers containing two different peptide-amphiphiles can be preparedas follows. Peptide-amphiphile 21 was dissolved in water at pH 7 at aconcentration of 5 mg/ml. Peptide-amphiphile 22 was dissolved in waterat pH 7 at a concentration of 5 mg/ml in a separate vial. Both solutionswere slightly cloudy. Upon mixing the two solutions the material formeda gel in less then one second.

Example 6

Several PAs with basic amino acids that can self-assemble at alkaline pHand also mixed systems, in which oppositely charged PAs self-assemble atneutral pH, were prepared. For this purpose PA compounds 22-25 wereprepared by standard solid phase peptide synthesis followed byalkylation with the C16 fatty acid, palmitic acid. See Table 2, FIG. 10b (PA 22) and FIGS. 10 c-e (PAs 23-25). The peptides thus prepared werecharacterized by electrospray mass spectroscopy and found to correspondto their respective expected masses. All four of the PAs were found todissolve in water at neutral pH at concentrations of 1 mg/ml or less.Molecules 22 and 23 were fully reduced to eliminate disulfide bondcrosslinking then used under anaerobic conditions or left in an excessof dithiothreitol. Compounds 24 and 25 could be dissolved at higherconcentrations (10 mg/ml) but molecules 22 and 24 were cloudy andviscous at this concentration.

Example 7

Solutions of each compound of example 6 were prepared at a concentrationof 0.1 mg/ml at neutral pH. Molecule 25 was slowly acidified (HCl) andfound to precipitate below a pH of 4.5 while increasing the solution'spH (KOH) left the molecule completely dissolved. Conversely, base wasslowly added to a solution of molecule 24 and it was found to be solubleuntil a pH above 9.5 was reached at which point a precipitate formed. Atneutral pH both molecules were completely dissolved, however, uponmixing these clear solutions a precipitation formed within seconds withno significant change of pH. At higher concentrations (5 mg/ml) mixingthe oppositely charged amphiphiles caused the immediate formation of abirefringent gel. Molecules 22 and 23, which can be covalentlycrosslinked through oxidation of their cysteine residues, behaved in asimilar fashion.

Example 8

Samples of the precipitated material in each of the six solutions ofexamples 6-7, one for each individual PA compound and mixed samples ofPAs 24 and 25 and PAs 22 and 23, were examined by negative staintransmission electron microscopy (TEM) and FT-IR. TEM revealed that inall cases the PA had self-assembled into nanofibers with nearly uniformdiameters of 7 nm+/−1 nm, often many microns long.

Example 9

FT-IR spectra of the preceding solutions showed strong hydrogen bondingbased on N—H stretching frequencies between 3276 and 3289 cm⁻¹, and allspectra showed significant parallel β sheet character based on theposition of the amide I band at 1630 cm⁻¹. Additional contributions inthis region between 1650 and 1680 cm⁻¹ indicate that the peptide regionalso adopts significant α-helix and random coil characteristics. The pHresponse of the PA compounds, their structure by TEM, and their IRspectra suggest, without limitation, a possible model of self-assembly:at neutral pH molecule 25 has a net negative charge of −1. This chargehelps to keep the molecule solubilized through electrostatic repulsionof other negatively charged species despite the large hydrophobic bulkof its fatty acid tail. Similarly, molecule 24 has a net charge of +2 atneutral pH and is soluble. Self-assembly is initiated when these chargesare eliminated, as in the pH driven self-assembly, or when the chargesare attractive instead of repulsive as in the case when oppositelycharged amphiphiles are mixed. The fact that the mixed self-assemblyoccurs at neutral pH where the individual molecules are soluble stronglysuggests that the self-assembly is driven by an electrostatic attractioninvolving both positively and negatively charged molecules and notsimply hydrophobic collapse involving one or the other PA.

Example 10

100 μl of 10 mg/ml solution of peptide-amphiphile 20 was treated with 1MCaCl₂ (adjusted to pH 6) drop wise in 1 μl increments. The solutionswere shaken after each addition of the CaCl₂ solution in order to obtainbetter diffusion of the divalent metal ions. Gelation was immediate.When examined by positive stain TEM the gel formed with calcium ions wasfound to be composed of nanofibers with the same dimensions as thoseformed by acid induced self-assembly and by surface drying. This calciuminduced self-assembly may be particularly useful for medicalapplications where formation of a gel at physiological pH is desired.Trivalent metal cations can also be used for gelation.

Example 11

To demonstrate cross-linking, in accordance with this invention, thegels formed above (examples 5 and 6) were treated with 0.05M I₂ whichwas adjusted to a pH of 3.5. The iodine solution was placed on top ofthe gel and allowed to slowly diffuse into the gel. After the iodinecolor had completely penetrated the gel, excess iodine was removed. Thegel was then soaked in a bath of deionized water which was periodicallychanged until the discoloration from the iodine was gone as judged byeye (roughly 48 hours depending on the size of the gel).

Example 12

Illustrating another gelation factor of this invention, self-assemblycan occur by simply taking a PA (such as PA 17, Table 2) or acombination of PAs dissolved in water at pH 8 and placing it on asurface which is allowed to dry (for example, directly on a carboncoated TEM grid). Upon examination of this preparation by negative stainTEM we observed clearly the formation of nanofibers. (See, FIG. 11.)

Example 13

Samples of the peptide-amphiphiles were prepared for TEM analysis in twodifferent ways. In some cases a small sample of the gel, prepared inbulk as described above, was smeared onto a holey carbon coated TEM grid(Quantifoil). Other samples were prepared directly on the grid byplacing 10 ul of 0.01-0.02% solution of PA directly on the grid. Thegrid was then placed into a sealed chamber with HCl vapors for 10minutes after which the grids were washed with de-ionized water. Tworoutine staining techniques, negative staining with PTA (phosphotungsticacid) or positive staining with uranyl acetate, were used in this study[Harris, 1991]. In all cases electron microscopy was performed at anaccelerating voltage of 200 kV. (See, FIGS. 10-13).

Example 14

To investigate the mineralization properties of PA nanofibers of thisinvention, the material was assembled and mineralized directly on aholey carbon coated TEM grid—to allow study of the dynamics of themineralization process while minimizing artifacts of TEM samplepreparation. To prepare samples, a drop of aqueous PA (1 mg/ml) wasmounted on the holey grid and the self-assembly of the PA was induced inan atmosphere of HCl vapor. The grids were immersed in aqueous iodine tooxidize the cysteine thiol groups to disulfides. After rinsing withdistilled water, the PA coated grids were treated with 5 μl of 10 mMCaCl₂ on one side and 5 μl of 5 mM Na₂HPO₄ on the other side. The twosolutions are able to mix only by passing through the holes in thecarbon support. Grids were examined by TEM at different time intervals(FIG. 3) and after 10 minutes inorganic material was observed to beconcentrated around the fibers thus increasing their contrast (acrystalline phase was not detected at this time by electrondiffraction). At 20 minutes the fibers begin to be covered withcrystalline mineral, although significant amourphous material remains.After 30 minutes plate shaped polycrystalline mineral is visiblethroughout the surface of the fibers (FIG. 3C and FIG. 5). The mineralwas analyzed by EDS (Energy Dispersion X-Ray Fluorescence Spectroscopy)which revealed a Ca/P ratio of 1.67.+−.0.08 which is consistent with theformation of HA with a formula of Ca₁₀(PO₄)₆(OH)₂ (FIG. 3 f).

Example 15

As controls for the experiment of example 6, carbon coated TEM gridswere treated as above but without PA fibers. In this case no mineraldeposit was found on the grids. In a second control, a PA was preparedin which phosphoserine was replaced by serine and treated as above withcalcium and phosphate. The nonphosphorylated fibers were observed by TEMafter 20 and 30 minutes of incubation and in both cases an amorphousmineral deposit around the fibers was observed, but crystals did notform within this time frame (FIG. 6).

Example 16

In order to discern the relative orientation of the HA crystals withrespect to PA fibers, several samples of example 6, in which isolatedmineralized bundles of PA fibers could be observed, were analyzed byelectron diffraction. In all cases preferential alignment of the HAcrystallographic c-axis with the PA fiber long axis was observed. (FIG.3D,E).

Example 17

Mineralization experiments show that PA fibers of this invention areable to nucleate hydroxyapatite on their surfaces. Negatively chargedsurfaces can promote mineralization by establishing local ionsupersaturation (S. Weiner, L. Addadi, J. Mater. Chem. 7, 689-702(1997)). Particularly, the two acidic aminoacids phosphoserine andaspartic acid used here are abundant in the proteins of mineralizedtissues proven to initiate crystal growth (L. Addadi, S. Weiner, Proc.Nail. Acad. Sci. 82, 4110-4114 (1985); G. Falini, S. Albeck, S. Weiner,L. Addadi, Science 271, 67-69 (1996); A. George, L. Bannon, B. Sabsay,J. W. Dillon, J. Malone, A. Veis, N. A. Jenkins, D. J. Gilbert, N. G.Copeland, J. Biol. Chem. 271, 32869-32873 (1996); S. Weiner, L. Addadi,J. Mater. Chem. 7, 689-702 (1997)). The fact that the fibers gain extraelectron density prior to formation of the crystalline phase suggeststhe above mechanism may be utilized in our system. More surprising isthe observation that the c-axes of the HA crystals are co-aligned withlong axes of the fibers (FIG. 4). This fact implies that the orientationof crystalline nuclei and the subsequent crystal growth are not randombut are controlled by the PA micelles. The exact mechanism of thiscontrol is not clear, however in similar systems such control is gainedby specific arrangement of acidic groups that promote growth of thecrystals in a particular orientation by an epitaxial mechanism (S. Mann,J. P. Hannington, R. J. P. Williams, Nature 324, 565-567 (1986); S.Weiner, L. Addadi, J. Mater. Chem. 7, 689-702 (1997); J. Aizenberg, A.J. Black, G. M. Whitesides, Nature 398, 495-498 (1999)). An analogousmechanism may be employed with PA fibers. Previous in vitro studiesshowing oriented crystal growth from organic templates were done mainlyin two dimensional systems. The results of this example show for thefirst time oriented crystal growth in a synthetic fibrous organicsubstrate.

Example 18

Several additional representative peptide-amphiphiles of this inventionwere prepared by manual solid phase peptide synthesis starting from 0.5mmoles of an FMOC-Asp(tBu)-WANG resin. Deprotection of the initial andsubsequent FMOC groups and was accomplished by double treatment with 15ml of 30% piperidine in DMF for 2 minutes and 7 minutes. The resin wasthen washed with 15 ml of DMF 5 times. With the exception of cysteinederivatives, amino acids (4 equivalents) were preactivated with HBTU(3.95 equivalents) and DiEA (6 equivalents) in 10 ml of DMF for twominutes. The activated solution was then added to the deprotected resinand allowed to shake for 30 minutes after which a small sample wasremoved and analyzed with ninhydrin to test for completeness of thereaction. Failed reactions were recoupled in an identical fashion.Cysteine was coupled via the FMOC-Cys(Trt)-OPfp activated ester with theaddition of 1 equivalent of DiEA in order to avoid suspectedepimerization found using the standard FMOC-Cys(ACM)-OH derivative withthe conditions described above. Other amino acid derivatives usedincluded FMOC-Gly-OH, FMOC-Arg(Pbf)-OH and FMOC-Ser(PO(OBzl)OH)—OH.Analogous derivatives available in the art may be used to couple A, S,L, K and other such residues, including those shown in Tables 1 and 2.The corresponding protecting groups and associated chemistries are knownin the art and may vary depending upon the subject amino acid residue.The final step was coupling of a fatty acid (palmitic acid) and wasaccomplished using 2 equivalents of the acid activated with 2 equivalentof HBTU and 3 equivalents of DiEA in 15 ml DMF for 2 minutes. Thesolution was allowed to react with the resin overnight. Likewise, otherreagents and starting materials of the sort useful in the preparation ofthe inventive compositions, but not limited to those shown in Tables 1and 2, are readily-available and known to those skilled in the art, suchreagents/starting materials as may be used for thehydrophobic/hydrocarbon and peptide (residues/monomers) components.

Cleavage and deprotection of the peptide-amphiphiles was done with amixture of TFA, water, triisopropyl silane (TIS) and ethane dithiol(EDT) in a ratio of 91:3:3:3 for three hours at room temperature. Thecleavage mixture and TFA washings were filtered into a round bottomflask. The solution was roto-evaporated and then redissolved in aminimum of neat TFA. This solution was triturated with colddiethylether. The white precipitation was collected by filtration anddried under vacuum.

Example 19a

To determine the relative number of metal ions necessary for gelation ofthe PAs, titration experiments were performed with PA molecules 26 and27. These tests show that the minimal amount of Ca⁺² and Gd⁺³ ionsneeded for the gelation is or about equal to the number of PA moleculesin solution. (See Table 3, below). PAs positively charged withoutcontaining negatively charged functional groups did not gel uponaddition of metal salts. None of the negatively chargedpeptide-amphiphiles except molecule 32 formed gels in the presence ofKCl at a ratio 1 molecule of PA per 20 molecules of KCl. Further testson the gelation abilities of monovalent salts showed that 10 mMsolutions of compounds 26 and 27 did not gel in the presence of KCl orNaCl at concentrations up to 6M. In contrast to other PAs, an additionof 200 mM of KCl to 10 mM solution of molecules 32 and 33 causedformation of a self-supporting gel.

Example 19b

An additional factor in self-assembly may be exemplified by thestructure of PAs 32 and 33. Both these molecules contain the IKVAV (SEQID NO: 1) sequence at the c-terminus of the peptide segment. Thissequence is comprised is of alternating extremely hydrophobic aminoacids I and V and more hydrophilic ones such as A, and K. Since the sidechains of adjacent amino acids are located on opposite sides of thepeptide backbone this sequence is, itself, amphiphilic. As such, PAs 32and 33 may be considered as double or two dimensional amphiphiles, withone axis of amphiphilicity coinciding with the backbone of the moleculeand amphiphilic peptide segment at the c-terminus, in addition tohydrophobic interactions between the alkyl moieties of the molecules.

Example 19c

The gels formed by addition of metal ions are remarkably stable. Suchexperiments show that they endure very basic conditions up to pH 11, andare stable at pH as low as 4. The gels also survive heating up to 100°C., though they slightly shrink. The gels also remain intact whenexposed to a volume of deionized water 10 times greater than the volumeof the gel for several days without any visible changes.

Example 20a

When Cu(ClO₄)₂ was added to the PA solutions the samples changed fromtransparent to blue upon gelation. The aqueous solution of copperperchlorate at same concentration was completely transparent. UV-Visanalysis of the gels of molecules 27 and 28 shows significant shift inabsorbance compare to the aqueous solution of Cu(ClO₄)₂ of sameconcentration. These results are suggestive of formation of metal PAcomplexes upon gelation.

Example 20b

A significant absorbance shift in the ultraviolet-visible spectroscopy(UV-vis spectra) of Cu⁺² induced gels, compared to aqueous solution ofCu(ClO₄)₂, implies formation of the metal-PA complexes. Thedisappearance of the peak at 1730 cm⁻¹ accompanied by decreasing of thedepth of minimum between Amide I and Amide II bands in the spectra ofmetal induced gels hints on the formation of metal-carboxyl ionic bonds.Also, the strength of the polyvalent metal ion induced gels incomparison to the pH driven self-assemblies under wide variety ofconditions suggests presence of relatively strong bonds between PAmolecules.

TABLE 3 Peptide-amphiphiles and their gelation properties. PA KCl***MgCl₂ CaCl₂ BaCl₂ Cu(ClO₄)₂ ZnBr₂ GdCl₃ SEQ ID NO: 26Alkyl*-C₄G₃S^((P))RGD-COOH Liquid weak gel gel no data gel gel 2  (−3)**gel 27 Alkyl-A₄G₃S^((P))RGD-COOH Liquid weak gel gel gel gel gel 9 (−3)gel 28 Alkyl-A₄G₃S^((P))KGE-COOH Liquid Viscous gel gel no data gel gel12 (−3) liquid 29 Alkyl-C₄G₃SRGD-COOH Liquid Cloudy gel gel no data gelgel 15 (−1) liquid 30 Alkyl-A₃G₂EQS—COOH Liquid gel gel gel gel gel gel24 (−2) 31 Alkyl-A₄G₃ERGDS—COOH liquid Cloudy cloudy cloudy no data gelgel 25 (−2) liquid liquid liquid 32 Alkyl-C₄G₃EIKVAV—COOH gel gel gel nodata no data gel gel 16 (−1) 33 Alkyl-C₄G₃KIKVAV—NH₂ weak Viscousviscous viscous no data viscous viscous 21 (+2) gel liquid liquid liquidliquid liquid *C₁₆ alkyl moieties (as tested) or about C₆ - about C₂₆ asreferenced elsewhere herein. **Overall charge of the molecule. ***KClconcentration was 200 mM for all molecules except molecules 26 and 27that were examined at KCl concentration up to 6 M. Other saltsconcentrations were 20 mM. Concentration of the peptide amphiphiles inall cases was 10 mg/ml (roughly 8 mM).

Example 21

One potential application of the peptide-amphiphile self-assembled gelsis in the area of tissue engineering, in particular the formation ofartificial extracellular matrices and cell delivery systems. The resultsof this example show PA self-assembly in situ and in vitro cell culturesystems. Body fluids as well as cell culture media contain significantamounts inorganic cations. It was presumed that the peptide amphiphileswould form a gel upon mixing with these liquids. Indeed, gel formationwas observed when 10 mM solutions of PAs 27, 28 or 33 in water weremixed with equal amounts of cell culture media. Solutions of PA 27 and28 were mixed with PBS and Hank's solutions depleted by Ca²⁺ and Mg²⁺,and no gel formation was observed—showing that multivalent metal ionspresent in the cell culture media induce self-assembly of negativelycharged PAs in a cell culture medium.

Example 22a

TEM studies revealed the ultrastructural organization of metal ioninduced PA gels. The electron micrographs of positively stained samplesand resin embedded section show that the gels are comprised of3-dimensional network of fibrils 5-6 nm in diameter, consistent withother measurements of dehydrated nanofibers (hydrated fibrils can be˜7.6 nm in diameter). The analysis of TEM data of positively stainedmaterial reveals that the uranyl acetate stains only peripheral parts ofthe fibrils, whereas the core remains unstained. The TEM of the resinembedded gel sections shows the same organization. It is apparent fromthe micrographs that the fibrils sectioned transversely have a doughnutappearance, with unstained central part and intensively stained outercircle. Since the uranyl acetate stains mainly charged groups and doesnot react with saturated hydrocarbons, these TEM results suggest that inthe presence of polyvalent metal ions PA assemble into cylindricalmicelles with their aliphatic tails in the core and the peptide partscomprising exterior layer. This structural organization of the PAnanofibers is similar to nanofibers assembled by a pH induced mechanism.

Example 22b

For TEM studies, positively stained samples of the PA gels were preparedas follows: The small amounts of the gels were mounted on the TEM gridand briefly dipped into deionized water in order to remove the excess ofthe material. The grids then were blotted against filter paper andplaced on droplets of 2% uranyl acetate (EMS) aqueous solution. Thesamples were stained for 30-40 min in the dark. The grids then werewashed in deionized water and dried. The samples were studied in theHitachi 8100 high resolution TEM at acceleration voltage 200 kV.

For resin embedding, the gels were fixed in 2% glutar aldehyde aqueoussolution (EMS). Samples then were washed in deionized water and stainedwith 2% aqueous uranyl acetate. The samples were dehydrated in ethanolgradient followed by propylene oxide. The samples then were transferredinto 1:1 mixture of propylene oxide and epoxy resin (SPIpon, SPI). After1 day of incubation the samples were transferred into pure epoxy resin.The resin was changed twice in 6 hours, The samples were polymerized fora day at 50° C. then at 60° C. for another day and at 70° C. for twomore days. Thin sections of the samples were cut using Leica Ultracut.The samples were studied in the JEOL 100C TEM at acceleration voltage100 kV.

Example 23a

FT-IR spectroscopy was employed to analyze interactions between the PAmolecules in the fibrils. The Amide I band maxima of the PA gel samples,prepared by addition of metal ions, was located at 1630-1640 cm⁻¹. Theposition of Amide I band maximum in this region suggests that thepeptide segments of the molecules adopt β-sheet conformation. In all thespectra studied no secondary peak at 1690 cm⁻¹, characteristic for theanti-parallel structures, has been detected, suggesting that thepeptides in the cylindrical micelles form parallel β-sheets—consistentwith a model based on TEM data. Amide A stretching band maximum in allspectra studied appears around 3280 cm⁻¹, indicating a high level ofhydrogen bonding in the PA supramolecular assemblies.

The spectra of the PA gels formed by pH induced self-assembly contain apeak in the 1720-1750 cm−1 region—characteristic of C═O stretching ofnon ionized carboxyls. In contrast, no evident peak has been detected inthis region in the spectra of the polyvalent metal ion induced gels. Ithas been reported in the literature that the formation of the metalcarboxyl complexes of amino acids results in a low frequency shift ofthis band maximum to the 1560 cm⁻¹-1610 cm⁻¹ region. No such peaks wereobserved in this region in the spectra of ion induced PA supramolecularassemblies, which may be due to the overlap between this peak andneighboring Amide I and Amide II bands. However the fact that the minimabetween Amide I and Amide II peaks is deeper in the spectra of pHinduced self-assemblies compare to ion induced self-assemblies suggestspresence of the peak in this region. (Reference is made to FIG. 15.)

Example 23b

For FT-IR studies the gels were quick frozen in liquid N2 andlyophilized. The KBr pellets of the lyophilized gels were analyzed on anFT-IR Biorad spectrometer. 32 scans per spectrum were taken atresolution 2 cm−1. The spectra were analyzed using Origin 6.0 program.

Example 24

The synthesis of the peptide amphiphiles illustrates in examples 19-22was performed using a standard synthesis scheme described elsewhere,herein, and as further available in the literature. Regarding gelationand with reference to the data of Table 3, 200 μm volumes of PAsolutions at concentrations 10 mg/ml (approximately 8 mM), pH 7.5 weremixed with 1 M solutions of NaCl, KCl, MgCl₂, CaCl₂, BaCl₂, ZnBr₂,CdCl₃, Ge and Cu(ClO₄)₂. The amount of polyvalent ions added to thesolutions varied from 0.5 to 5 metal ions per molecule of PA. Formonovalent metal ions the maximum concentration of 6 M of metal ions per10 mM of PA molecules has been tested. The solutions of the metal saltswere added to the PA solutions by micropeppetors, the solutions werestirred briefly, in order to obtain better mixing of the components. Theformation of self-supporting gels was examined by flipping of the vialsup side down. In order to test the ability of cell compatible solutionsto induce a gelation of PAs equal amounts 10 mM PA solutions were mixedwith following formulations: MEM-α, DMEM containing 10% of fetal bovineserum, PBS without Ca⁺² and Mg⁺² and Hank's solution without Ca⁺² andMg⁺² (Gibco). The ability of the above solutions to induce formation ofthe PAs was examined visually by flipping the vials up side down.

Various other amphiphile compositions of this invention can be preparedin analogous fashion, as would be known to those skilled in the art andaware thereof, using known procedures and synthetic techniques orstraight-forward modifications thereof depending upon a desiredamphiphile composition or peptide sequence.

As demonstrated by the preceding examples, figures and data, the factthat the hydroxyapatite crystals grow on the bundles of fibers withtheir c-axes oriented along the long axes of the micelles could be ofinterest for design of new materials for mineralized tissue repair. Thisnanoscale organization resembles that of hydroxyapatite crystals inmineralized ECM in which the HA crystals also grow in parallel arrayswith their c-axes co-aligned with long axes of the organic fibers (W.Traub, S. Weiner, Proc. Nat. Acad. Sci. 86, 9822-9826 (1989)). Thisarrangement is the most important characteristic of the biomineralsbelonging to the bone family (S. Weiner, H. D. Wagner, Annu. Rev. Mater.Sci. 28, 271-298 (1998)). The organization of the collagen fibers,porosity, mineral-organic ratio vary in different members of thisfamily, yet all of them are built from the collagen fibrils containingparallel arrays of hydroxyapatite crystals (W. Traub, S. Weiner, Proc.Nat. Acad. Sci. 86, 9822-9826 (1989); S. Weiner, W. Traub, FASEB J. 6,879-885 (1992); W. J. Landis, K. J. Hodgens, J. Arena, M. J. Song, B. F.McEwen, Microsc. Res. Techniq. 33, 192-202 (1996)).

It has been proposed that in the case of the pH triggered self-assemblycylindrical micelles form due to the protonation of acidic groups of thepeptide and reduction of the repulsive forces between molecules—that inthe case of pH triggered self-assembly of the PAs the major drivingforce is hydrophobic interactions between the aliphatic tails of themolecules, rather the interactions between the peptide segments. Theresults reported herein show that the interactions between peptidesegments of the PA via metal bridges or due to amphiphilicity of thesequence, may also play a role in the self-assembly. Gelation underphysiological conditions demonstrate such PAs as potentially importantmaterials for biomedical applications, such as tissue engineering ordelivery of cells, drugs or other therapeutic agents. Supramolecularassemblies induced by addition of metal ions are stable in wide pH rangeand provide as potential applications embedding cells in a gel or insitu gelation of a tissue repair material.

While the principles of this invention have been described in connectionwith specific embodiments, it should be understood clearly that thesedescriptions are added only by way of example and are not intended tolimit, in any way, the scope of this invention. For instance, variouspeptide amphiphiles have been described in conjunction with specificresidues and corresponding cell adhesion, but other residues can be usedherewith to promote a particular cell adhesion and tissue growth on thenanostructures prepared therefrom. Likewise, while the present inventionhas been described as applicable to biomimetic material or tissueengineering, it is also contemplated that gels or related systems ofsuch peptide amphiphiles can be used as a delivery platform or carrierfor drugs, cells or other cellular or therapeutic material incorporatedtherein. Other advantages and features will become apparent from theclaims filed hereafter, with the scope of such claims to be determinedby their reasonable equivalents, as would be understood by those skilledin the art.

1. A method of making a cylindrical micellar nanofiber composed ofbeta-sheets of peptide segment of peptide amphiphile comprising thesteps of: (a) providing the peptide amphiphile molecule in an aqueoussolution, wherein said peptide amphiphile molecule comprises a single C₆to C₂₂ linear hydrocarbon segment covalently linked to a peptide segmentcomprising a bioactive epitope sequence selected from RGD, KGE, IKVAV(SEQ ID NO: 1) and YIGSR (SEQ ID NO: 20); and (b) contacting saidpeptide amphiphile molecule with an effective amount of a metal ionsufficient to induce formation of a cylindrical micellar nanofibercomposed of beta-sheets.
 2. The method of claim 1, wherein saidcontacting step occurs under physiological conditions.
 3. The method ofclaim 1, wherein said metal ion is present in a bodily fluid.
 4. Themethod of claim 1, wherein the metal ion selected from the groupconsisting of Ca²⁺, Mg²⁺, Cd²⁺, Fe²⁺ and Zn²⁺ divalent ions, Al³⁺, Fe³⁺,and Gd³⁺ trivalent ions, and combination of said ions.
 5. The method ofclaim 1, wherein said peptide amphiphile molecule has a net charge atsubstantially physiological pH.
 6. The method of claim 1, wherein saidpeptide segment further comprises at least one phosphorylated residue.7. The method of claim 6, wherein said phosphorylated residue isphosphoserine.
 8. The method of claim 1, wherein said peptide segmentfurther comprises a residue with a functional moiety capable ofintermolecular covalent bond formation.
 9. The method of claim 8,wherein said residue is cysteine.
 10. The method of claim 9, whereinsaid peptide segment further comprises at least one phosphoserineresidue.
 11. The method of claim 10, wherein said peptide segmentfurther comprises at least one glycine residue between said cysteine andphosphoserine residues.
 12. The method of claim 1, wherein said peptidesegment comprises a first peptide sequence selected from the groupconsisting of CCCCGGGS(p) (SEQ ID NO: 3), CCCCGGGS (SEQ ID NO: 26),CCCCGGGE (SEQ ID NO: 27), AAAAGGGS(p) (SEQ ID NO: 28), AAAAGGGS (SEQ IDNO: 29), GGGS(p) (SEQ ID NO: 30), and GGGS (SEQ ID NO: 31) and a secondpeptide sequence selected from RGD, KGE, IKVAV (SEQ ID NO: 1) and YIGSR(SEQ ID NO: 20), wherein S(p) stands for phosphorylated serine residue.13. The method of claim 1, wherein said hydrocarbon segment is linked tothe N-terminus of said peptide segment.
 14. The method of claim 1,wherein said cylindrical micellar nanofiber has dimensional andfunctional characteristics mimetic of collagen fibrils.
 15. The methodof claim 1, wherein said cylindrical micellar nanofiber forms a conicalshape comprised of a narrow hydrocarbon tail at one end and a bulkierpeptide region at the other.
 16. The method of claim 1, wherein saidcylindrical micellar nanofiber displays a secondary structure thatincludes both beta-sheets and alpha-helices.